Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 January 2012 to 24 January 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection 2011-07-19 to 2011-07-21; Date of signature 2011-08-31
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 9-decenoic acid, methyl ester (9DAME)
- Physical state: Clear colourless liquid
- Analytical purity: 99%
- Lot/batch No.: 184-109
- Date received: 2011-04-01
- Storage conditions: room temperature in the dark under nitrogen

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: 2014C Teklad Global Rodent diet available ad libitum
- Water: Tap water available ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °C
- Humidity: 30 - 70%
- Air changes: 15 changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
This vehicle was chosen as it produced the most suitable formulation at the required concentration.
Concentration:
25%, 50% and 100% v/v in acetone/olive oil 4:1.
No. of animals per dose:
4 animals per dose
Details on study design:
RANGE FINDING TESTS:
- A preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the undiluted test item to the dorsal surface of each ear for three consecutive days.
- Irritation: Very slight erythema was noted on both ears on Days 2 and 3. No signs of systemic toxicity or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:
- The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
- A further group of four mice received the vehicle alone in the same manner.
- Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmol) giving a total of 20 μCi to each mouse.
- Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
- Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The stimulation index was 4.05. Therefore, α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows: - Concentration (% v/v) in acetone/ olive oil 4:1, Vehicle: n/a - Concentration (% v/v) in acetone/ olive oil 4:1, 25 %: 1.61 - Concentration (% v/v) in acetone/ olive oil 4:1, 50 %: 3.02 - Concentration (% v/v) in acetone/ olive oil 4:1, 100 %: 2.42
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
The radioactive disintegrations per minute per lymph node are as follows: - Concentration (% v/v) in acetone/ olive oil 4:1, Vehicle: 12665.97 - Concentration (% v/v) in acetone/ olive oil 4:1, 25 %: 20382.35 - Concentration (% v/v) in acetone/ olive oil 4:1, 50 %: 38258.42 - Concentration (% v/v) in acetone/ olive oil 4:1, 100 %: 30617.01

Any other information on results incl. tables

An expert review of these study results was provided by Dr. David Basketter. Dr. Basketter's expert rerpot is attached.

CLINICAL OBSERVATIONS AND MORTALITY DATA

Mild redness on the ears and neck was noted, post Days 2 and 3, in animals treated with the undiluted test item at a concentration 50 % v/v in acetone/olive oil 4:1. There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

BODYWEIGHT

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Table 1: Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (% v/v) in acetone/olive oil 4:1

dpm

Dpm/ Nodea

Stimulation Indexb

Vehicle

12665.97

1583.25

n/a

25

20382.35

2547.79

1.61

50

38258.42

4782.30

3.02

100

30617.01

3827.13

2.42

dpm = Disintegrations per minute

a= Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8

(total number of lymph nodes)

b= Stimulation Index of 3.0 or greater indicates a positive result

na = Not applicable

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item returned a borderline response under the conditions of the test. An expert review of these study results (Basketter, 2012) indicated that 9-decenoic acid, methyl ester (9DAME) does not contain any structural alerts for reactivity associated with skin sensitisation and is a long chain unsaturated fatty acid. Published work has indicated that unsaturated fatty acids have the potential to generate false positive results in the LLNA. Several long chain unsaturated fatty acids, such as oleic acid, have proven to be positive in the LLNA but were clear negatives in guinea pig maximisation test conducted in parallel, an outcome which was consistent with their absence of skin sensitising effects in humans.

Based upon the above considerations, the very borderline nature of the single positive result and the absence of a dose response in this test it is feasible that a false positive result may have been obtained and hence 9-decenoic acid, methyl ester (9DAME) is not considered to be a skin sensitiser.